EP0624645B1 - Menschliches Chondromodulin-I-Protein - Google Patents

Menschliches Chondromodulin-I-Protein Download PDF

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EP0624645B1
EP0624645B1 EP94107364A EP94107364A EP0624645B1 EP 0624645 B1 EP0624645 B1 EP 0624645B1 EP 94107364 A EP94107364 A EP 94107364A EP 94107364 A EP94107364 A EP 94107364A EP 0624645 B1 EP0624645 B1 EP 0624645B1
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protein
chondromodulin
dna
seq
sequence
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EP0624645A1 (de
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Fujio Suzuki
Yuji Hiraki
Kazuhiro Takahashi
Junko Suzuki
Jun Kondo
Atsuko Kohara
Akiko Mori
Ei Yamada
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Hiraki Yuji
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a novel human chondromodulin protein. More particularly, it relates to chondromodulin-I protein capable of stimulating the growth of chondrocytes in the presence or absence of fibroblast growth factor an isolated DNA (gene) encoding said protein, expression vectors containing said DNA, transformants capable of producing recombinant chondromodulin-I protein, a process for producing chondromodulin-I protein by culturing said transformants and a pharmaceutical composition containing chondromodulin-I protein as an active ingredient.
  • the present invention also relates to the use of chondromodulin-I protein in the treatment of fracture and various cartilage diseases and as an anti-tumor drug.
  • chondromodulin protein The protein which concerns the above-mentioned growth of chondrocytes is known as chondromodulin protein having biological activities as illustrated below.
  • chondrocytes plays an important role in the course of recovery from fracture or various cartilage diseases as follows: inflammatory reaction at the injured site, growth of the periost-derived cells, expression and growth of chondrocytes, synthesis of extra-cellular ground substances, calcification of said substances, and replacement thereof with bone tissues.
  • the growth of cartilage tissue at the site of fracture is essential for the formation of bone tissue.
  • the growth of the chondrocytes is also important in the course of the recovery from cartilage diseases accompanied by cartilage destruction or injury.
  • infiltration of blood vessels into tissues occurs for the supply of necessary energy to tumor cells, and therefor the inhibition of such a infiltration is thought to be effective for the prevention of growth or metastasis of tumor cells.
  • chondromodulin-I protein has been cloned from a cDNA library constructed from fetal bovine cartilage and expressed in animal cells.
  • the expressed recombinant protein possesses activities equivalent to those of purified bovine chondromodulin-I protein. Seki et al., Biochemical and Biophysical Research Communications, 175, 971-977 (1991) ; and European Patent Publication No. 473,080 .
  • the term "human chondromodulin-I protein” or "human chondromodulin-I” will be expressed by the abbreviation "hChM-I" in some cases.
  • chondromodulin protein capable of inhibiting the amplification of chondrocytes or infiltration of blood vessels, said protein being less antigenic in human, and thereby providing treating methods effective on the above-mentioned diseases.
  • industrial production of said protein from cartilage tissue was extremely difficult and practical application of said protein has been hindered because of the lacking of means to obtain the protein in large amount.
  • the present inventors have studied extensively with the aim of producing a large amount of human chondromodulin protein by the use of recombinant DNA techniques and have now succeeded in the isolation of a novel protein, which belongs to a family of chondromodulin protein, useful for the establishment of the purpose of the invention, cloning of a gene (cDNA) encoding said protein, construction of an expression vector, transformation of heterogeneous cells, and production of recombinant protein by culturing the transformants.
  • the present inventors also investigated physiological activities of thus obtained human chondromodulin-I protein and demonstrated that said protein possesses above-mentioned activities and are useful in the treatment of fracture, various cartilage diseases, cancers and the like and can be formulated into pharmaceutical compositions.
  • this invention provides a human chondromodulin-I protein, which is a water-soluble protein composed by one polypeptide, has a molecular weight of about 26,000 dalton on SDS-polyacrylamide gel electrophoresis and has abilities to stimulate the growth of chondrocytes in the presence or absence of fibroblast growth factor, to promote the differential potency of chondrocytes and to inhibit the growth of vascular endothelial cells.
  • This invention also provides an isolated gene (DNA) encoding human chondromodulin-I protein and expression vectors containing sequences required for the expression of said gene, which comprise, at least, a promoter sequence, signal peptide-like sequence, DNA sequence encoding the chondromodulin-I protein, and a terminator sequence, if desired.
  • DNA isolated gene
  • sequences required for the expression of said gene comprise, at least, a promoter sequence, signal peptide-like sequence, DNA sequence encoding the chondromodulin-I protein, and a terminator sequence, if desired.
  • This invention further provides a transformant transformed by an expression vector of the invention and a process for producing human chondromodulin-I protein by culturing said transformation in an appropriate medium for the expression of the DNA encoding chondromodulin-I protein and recovering the chondromodulin-I protein from the resultant cultured broth.
  • This invention also provides the recombinant human chondromodulin-I protein products produced by the method of present the invention.
  • This invention also provides the use of human chondromodulin-I protein obtained according to the procedure of the invention in the treatment of fracture, various cartilage diseases and cancer.
  • the chondromodulin-I protein was obtained and purified by isolating chondrocytes from human cartilage, culturing the cells, separating the supernatant from cultured broth by centrifugation, concentrating the supernatant by ultrafiltration, subjecting the concentrate to a molecular sieve chromatography on Sephacryl S1200 column, and purifying the resultant product repeatedly with YMC pack C4 chromatography while changing the elution conditions.
  • chondromodulin-I protein can be obtained, as is described in Examples below, by culturing cells transformed with hChM-I gene, separating the chondromodulin-I protein associated with albumin from other contaminating proteins in the cultured broth by means of Blue Sepharose column or the like, and purifying repeatedly by, for example, chromatography using YMC pack C4 column or the like while changing eluting conditions.
  • purified protein of the invention has a molecular weight of about 26,000 dalton on SDS-PAGE and has activities to stimulate the growth of chondrocytes in the presence or absence of fibroblast growth factor (FGF), to promote the differential potency of chondrocytes, and to inhibit the growth of vascular endothelial cells.
  • FGF fibroblast growth factor
  • the amino acid sequence of the purified peptide was determined, which is provided in SEQ ID NO: 2, 3 or 4.
  • the present inventors then isolated cDNA encoding chondromodulin-I protein, cloned said DNA and constructed expression vectors.
  • a base sequence encoding the chondromodulin-I protein is provided in SEQ ID NO: 2, 3, 4, 5, 6 or 7.
  • SEQ ID NO: 2, 3 and 4 contain partly a base sequence(s) of bovine gene, as primers used in PCR have been made on the basis of bovine chondromodulin gene.
  • human chondromodulin-I protein refers to both of naturally occurring human chondromodulin-I protein and recombinant human chondromodulin-I protein produced by the method of the invention.
  • a DNA fragment(s) encoding chondromodulin-I protein of the invention can be obtained in conventional manners using DNA libraries containing a gene encoding the protein as illustrated below.
  • Examples of library usable are those prepared from RNA isolated from normal human cartilage or human chondrosarcoma, including plasmid cDNA library, phage cDNA library, phage genomic library.
  • phage cDNA library normal human cartilage or human chondrosarcoma tissue is pulverized in liquid nitrogen, homogenize in a solvent such as aqueous guanidium isothiocyatate solution, and separating precipitates of total RNA by cesium chloride equilibrium density gradient centrifugation according to the method of Chirgwin et al., Biochemistry, 18, 5294-5299 (1978) .
  • a solvent such as aqueous guanidium isothiocyatate solution
  • separating precipitates of total RNA by cesium chloride equilibrium density gradient centrifugation according to the method of Chirgwin et al., Biochemistry, 18, 5294-5299 (1978) .
  • the resultant total RNA is purified by phenol extraction and ethanol precipitation, it is further purified with chromatography using oligo(dT)cellulose column to isolate the objective poly(A)-containing mRNA (polyA + mRNA), i.e., mRNAs
  • Single-stranded cDNA can be obtained by hybridizing mRNAs previously prepared with DNA primers such as those described in Nature, 329, 836-838 (1987) , specifically, those consisting of DNAs shown in SEQ ID NO: 11 and 12, or with Oligo(dT) consisting of 12-18 deoxythymidines in the presence of reverse transcriptase.
  • the single-stranded cDNA is converted into double-stranded cDNA by treating with Escherichia coli DNA polymerase I or E . coli DNA ligase, RNase H or the like in a conventional manner, which is then blunt-ended with T4 DNA polymerase.
  • small DNA fragments such as Eco RI adapter with T4 DNA ligase to generate the same base sequences as those producible with the restriction enzyme.
  • a DNA having Eco RI restricted ends is also obtainable by treating the cDNA with methylase such as Eco RI methylase to protect inherent Eco RI restriction site(s), adding Eco RI linkers or the like to the both ends with T4 DNA ligase, and digesting with restriction enzyme Eco RI.
  • methylase such as Eco RI methylase
  • Eco RI linkers or the like to the both ends with T4 DNA ligase, and digesting with restriction enzyme Eco RI.
  • Bam HI restriction site
  • the series of procedures described above would be carried out using, for example, Bam HI adapter, or Bam HI methylase, Bam HI linker, and restriction enzyme Bam HI.
  • the cDNA strand having ends treated as mentioned above is then packaged into a commercially available ⁇ phage vector, for example, ⁇ ZAP (PROMEGA Biotechs, Inc.) or pGEM2 (PROMEGA Biotechs, Inc.) at Eco RI site in a conventional manner to obtain recombinant ⁇ phage DNAs or recombinant plasmid DNAs.
  • ⁇ ZAP PROMEGA Biotechs, Inc.
  • pGEM2 PROMEGA Biotechs, Inc.
  • the resultant ⁇ phage DNAs are used in the in vitro packaging by means of commercially available in vitro packaging kit such as Gigapack Gold (PROMEGA Biotechs, Inc.) to obtain ⁇ phage particles containing recombinant ⁇ phage DNA.
  • the resultant ⁇ phage particles are then transfected into host cells such as E . coli according to a conventional manner ( Molecular Cloning, Cold Spring Harbor Laboratory, p.85 (1982 )) for amplification.
  • Recombinant plasmid DNAs will be transformed into host cells such as E. coli in a conventional manner and transformants grown.
  • the amplified phages are transferred onto a nylon membranes such as gene screening plus or a nitrocellulose filter and treated with an alkali to remove protein to obtain ⁇ phage DNA or plasmid DNA containing cDNA.
  • the DNAs of cDNA clone are hybridized with 32 P-labeled probe, which has been prepared from DNA fragments of previously cloned bovine chondromodulin gene in a conventional manner or using commercially available kit, and selected by plaque hybridization according to the method described in Molecular Cloning, Cold Spring Harbor Laboratory, p.320-328 (1982 ) to yield complete or a part of cDNA encoding hChM-I.
  • a partial fragment of human chondromodulin-I gene can be obtained by polymerase chain reaction (PCR) using PCR primers designed on the basis of amino acid sequence of bovine chondromodulin-I protein.
  • PCR polymerase chain reaction
  • cDNA synthesized on the basis of RNA extracted from human chondrosarcoma or human normal chondrocyte in a conventional manner can be used.
  • a gene encoding entire sequence of human chondromodulin-I protein can be obtained by carrying out PCR repeatedly, by carrying out PCR with primers designed on the basis of a primer(s) used in the synthesis of template cDNA, or by carrying out PCR with primers based on appropriate anchor sequence attached at the 3' end of said cDNA.
  • a synthetic oligonucleotide prepared on the basis of DNA sequence deduced from the amino acid sequence of chondromodulin-I protein is also available.
  • DNA can be prepared from positive plaques after the amplification of phages as described ( T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 85 (1982 )), digested with an appropriate restriction enzyme such as Eco RI or the like, and subcloned into a plasmid such as pUC18, and the base sequence of desired cDNA segment can be determined by, for example, dideoxy method ( Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463, (1977 )).
  • base sequence for example, that shown in the SEQ ID NO: 2, 3, 4, 5, 6 or 7, is a fragment of 892 or 1006 nucleotides long in total and encodes a protein having 296 or 334 amino acids, wherein said protein contains the amino acid sequence of the mature protein.
  • Base sequences shown in SEQ ID NO: 2, 3 and 4 contains partly a base sequence(s) of bovine gene though, the amino acid sequences coded by them are in agreement with that of human chondromodulin-I protein.
  • chondromodulin-I protein As the DNA encoding chondromodulin-I protein was cloned and its sequence was determined in the present invention, one can easily construct expression vectors which enable host cells to produce chondromodulin-I protein using the known recombinant technology for example, by inserting into a known expression vector the DNA compound, after modification of 5' terminal at an appropriate site of the vector, downstream from a promoter, using a well known method per se, and introducing the expression vector harboring the cDNA into a host cell such as Escherichia coli cell, yeast cell, animal cell according to the method known to one of skill.
  • a host cell such as Escherichia coli cell, yeast cell, animal cell according to the method known to one of skill.
  • This invention can be accomplished using any expression vectors having a promoter at an appropriate site to make the DNA encoding chondromodulin-I protein expressed in a selected host cell.
  • human chondromodulin-I protein To accomplish the industrial production of human chondromodulin-I protein, it is necessary to construct a stable host-vector system which can express biologically active protein.
  • Factors that must be considered are: naturally-occurring human chondromodulin-I protein is a sugar protein; human chondromodulin-I molecule contains a lot of cysteine residues whose refolding affect greatly on the acquisition of physiological activity; and the expression product must be processed in living body (cells) to mature-type human chondromodulin-I protein. Taking into account these factors, animal cells are preferred as hosts for purposes of the present invention.
  • host cells usable for transformation include animal cells described in working Examples below. However, it is not restrictive and other host cells such as microorganisms or insect cells can be used.
  • Animal cells usable in the present invention are CHO cell, COS cell, mouse L cell, mouse C127 cell, mouse FM3A cell. These cells have advantage that they can produce and secrete mature-type hChM-I by introducing a gene encoding hChM-I in the form of precursor protein.
  • expression plasmids preferably contain SV40 promoter, metallothionein gene promoter or the like.
  • An expression vectors can be constructed by inserting hChM-I gene modified to have signal sequence from 5' terminal downstream from a promoter.
  • the expression vector may contain two or three hChM-I genes inserted in such a manner that genes are connected tandemly from 5' to 3' direction.
  • 2 - 3 genes each having a promoter such as SV40 promotor attached to its 5' site can be connected tandemly.
  • a polyadenylation site for example, one derived from SV40 DNA, ⁇ -globin gene or metallothionein gene is placed downstream from the hChM-I gene.
  • Expression vectors may contain a gene that serves as a marker for selection when transformed into animal cells such as CHO cell.
  • selectable marker are DHFR gene that gives methotrexate-resistance and 3'-deoxycistoleptamine antibiotic G-418 gene and the like.
  • a promoter such as SV40 promoter and a polyadenylation site, respectively. It can be accomplished by inserting a marker gene into hChM-I expression vector downstream from polyadenylation site of hChM-I gene.
  • Expression vectors may not contain selectable marker for transformants. In such a case, double-transformation will be carried out using hChM-I expression vector and a vector containing selectable marker in transformants such as pSV2neo, pSV2gpt, pMTVdhfr.
  • Animal cells transformed by the double-transformation can be selected on the basis of phenotype as mentioned above due to the expression of selectable marker. After host cells in which hChM-I has been expressed are detected, double-transformation can be repeatedly conducted using different selectable marker so as to increase the yield of expression product hChM-I.
  • Example of plasmid vector useable as an expression vector is pKCR ( Proc. Natl. Acad. Sci. USA, 78, 1528 (1981 )) containing SV40 early promoter, splicing sequence DNA derived from rabbit ⁇ -globin gene, polyadenylation site derived from rabbit ⁇ -globin gene, polyadenylation site derived from SV40 early promoter, and origin of transcription and ampicillin-resistant gene derived from pBR322.
  • expression vector is introduced into animal cells by transfection with calcium phosphate. Cultivation of transfectants can be carried out in a conventional manner by means of suspension culture or adhesion culture in a medium such as MEM, RPMI1640 in the presence of 5-10 % serum or appropriate amount of insulin, dexamethasone, transferrin, or in a serum-free medium. Animal cells expressing hChM-I are expected to secrete hChM-I in culture supernatant and therefore it is possible to carry out separation and purification of hChM-I using the supernatant of cultured broth of transformants. The culture supernatant containing hChM-I can be purified by means of chromatography using heparin sepharose, blue sepharose.
  • Expression vectors functional in microorganisms such as Escherichia coli , Bacillus subtilis will preferably comprise promoter, ribosome binding (SD) sequence, chondromodulin-I protein-encoding gene, transcription termination factor, and a regulator gene.
  • SD ribosome binding
  • promoters include those derived from Escherichia coli or phages such as tryptophane synthetase (trp), lactose operon (lac), ⁇ phage P L and P R , T 5 early gene P 25 , P 26 promoter. These promoter may have modified or designed sequence for each expression vector such as pac promoter ( Agr. Biol. Chem., 52: 983-988, 1988 ).
  • SD sequence may be derived from Escherichia coli or phage, a sequence which has been designed to contain a consensus sequence consisting of more than 4 bases, which is complementary to the sequence at the 3' terminal region of 16S ribosome RNA, may also be used.
  • the transcription termination factor is not essential. However, it is preferable that an expression vector contains a p-independent factor such as lipoprotein terminator, trp operon terminator.
  • these sequences required for the expression of the chondromodulin-I protein gene are located, in an appropriate expression plasmid, in the order of promoter, SD sequence, chondromodulin-I protein gene and transcription termination factor from 5' to 3' direction.
  • Typical examples of expression vector are pVAI2 ( Japanese Patent Laid-open No. 95798/1989 ) and commercially available pKK233-2 (Pharmacia).
  • pVAI2 Japanese Patent Laid-open No. 95798/1989
  • pKK233-2 commercially available pKK233-2
  • pGEK plasmids pGEK (Pharmacia)
  • pGEK plasmids
  • a suitable host cell can be transformed with an expression vector comprising the DNA encoding chondromodulin-I protein in a conventional manner to give a transformant.
  • the cultivation of the transformants can be carried out using any of the well known procedures in literatures such as Molecular Cloning (1982).
  • the cultivation is preferably conducted at a temperature from about 28°C to 42°C.
  • Expression vectors used for transforming other host cells consist of substantially the same elements as those described in the above. However, there are certain preferable factors as follows.
  • MAXBAC TM When insect cells are used, a commercially available kit, MAXBAC TM is employed according to the teaching of the supplier (MAXBAC TM BACULOVIRUS EXPRESSION SYSTEM MANUAL VERSION 1.4). In this case, it is desirable to make a modification to reduce the distance between the promoter of polyhedrin gene and the initiation codon so as to improve the expression of the gene.
  • chondromodulin protein produced by transformants can be conducted using any one of known procedures to one of skill in the art.
  • chondromodulin protein produced by transformants can be conducted using any one of known procedures to one of skill in the art.
  • the recombinant polypeptide expressed by the host cells such as microorganisms including E. coli, insect cells and animal cells can be recovered from the cultured broth by known methods and identified by, for example, immunoreactions between the expressed protein and a rabbit antiserum raised against a synthetic peptide containing a fragment of human chondromodulin-I protein, using a conventional method such as Western blot analysis.
  • Biological activities of chondromodulin protein can be determined according to the described method ( Suzuki, et al., Methods in Enzymology, 146; 313-320, 1987 ).
  • the chondromodulin protein-I was also evaluated about the inhibiting activity against vascular infiltration by determining the preventive effect on the growth of vascular endothelial cells.
  • the evaluation was based on the inhibition against [ 3 H]thymidine uptake by aortic endothelial cells as will be hereinafter described in detail in Examples.
  • the uptake of radioactive thymidine was apparently inhibited.
  • hChM-I for application of hChM-I to clinical treatment, it is usable alone or as a pharmaceutical composition formulated with pharmaceutically acceptable carriers therefor.
  • the content of hChM-I, an active ingredient, can be 1-90 %(w/w) related to carriers.
  • hChM-I of the present invention can be formulated as a medicine for external application and use in the treatment of fracture, cartilage disease, which medicine can be prepared by, for example, mixing with, impregnating into, or applying onto physiologically acceptable carriers including collagen, aterocollagen, gelatin, hyaluronic acid, polyethylene glycol, polylactose, bone cement, hydroxyapatite, ceramics, carbon fiber, fibrin starch and the like.
  • It can be orally administered after formulating into an appropriate form such as granules, fine granules, powders, tablets, hard capsules, soft capsules, syrups, emulsions, suspensions, solutions. It can be formulated into injectable forms to be administered intravenously, intramuscularly, topically, or subcutaneously, or into suppositories. These formulations for oral, intrarectal or parenteral administration can be prepared using organic/inorganic carriers/diluents in the form of solid/liquid. Examples of excipient usable in solid formulations include lactose, sucrose, starch, talc, cellulose, dextrin, kaolin, calcium carbonate.
  • Liquid formulations for oral administration may contain inert diluents commonly used in the art such as water, plant oil. Addition to the inert diluent, such a formulation can cantain additives, for example, humectant, suspending aides, sweetening agents, aromatics, coloring agents, preservatives.
  • the liquid formulations may be included in capsules made of absorbable substance.
  • solvent or suspending agent for parenteral formulations such as injections, suppositories include water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, lecithin.
  • bases for suppositories include cacao butter, emulsified cacao butter, laurin tallow, witepsol. Preparation of these formulations can be carried out in a conventional manner.
  • the clinical dose of hChM-I of the present invention varies depending on the manner of administration, age, weight, and the condition of patient to be treated.
  • Appropriate daily dosage of hChM-I of the present invention on oral or external administration to adult is generally about 1 ng - 50 mg (for injection, 1/10 or less than 1/10 of this dosage), which may be administered once, in two to several divisions at appropriate intervals, or intermittently. For injection, it is preferable to administer the above-mentioned dose continuously or intermittently.
  • hChM-I of the present invention may be in any form that can exert the biological and/or physiological activities of hChM-I, for example, purified hChM-I, recombinant hChM-I, cultured broth of transformants, separated transformants, treated transformants, immobilized transformants, crude enzyme solution, enzymatically-treated product.
  • Plasmid DNA was obtained from cloned bovine chondromodulin gene ( Seki et al., Biochemical and Biophysical Research Communications, 175, 971-977 (1991) ; and European Patent Publication No. 473,080 ) according to the method of T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 85-96 (1982 ).
  • a large amount of plasmid DNA containing bovine chondromodulin gene was recovered and purified from E. coli transformants harboring pUC19 vector containing at its Eco RI site an about 1.4 kb gene sequence of bovine chondromodulin gene encoding the whole protein.
  • the plasmid DNA (20 ⁇ g) was digested with restriction enzymes Eco RI (about 200 U) and Pst I (about 200 U) at 37°C for 2 hr and subjected to electrophoresis on agarose gel to separate vector DNA and bovine chondromodulin gene in a conventional manner.
  • the gel was stained with ethydium bromide in a conventional manner and gel containing DNA fragment corresponding to bovine chondromodulin gene was cut out under UV light.
  • the gel containing bovine chondromodulin gene was treated according to the method of Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, 164-170 ( 1982 ) to recover and purify DNA fragment (about 2 ⁇ g) for probe.
  • the DNA fragment (100 ng) was labeled with 32 P using RTC DNA Labeling Kit (Pharmacia, Inc.) according to the manufacture's instruction attached thereto.
  • the NZY soft-agar was prepared by autoclaving NZY medium containing 0.7% agar powder.
  • the plaque hybridization was carried out by transferring ⁇ phage clones in NZY soft-agar to nitrocellulose filter (BA85, S & S Inc.) by putting a filter on each plate and removing gently.
  • nitrocellulose membranes were put on soft-agar in each plate and removed to transfer phages. The both membranes were marked equally to show the relative positions on the plate. Each membrane was placed for 2 min on a filter paper previously soaked with 0.2 M sodium hydroxide/1.5 M sodium chloride. After removal of fluid with dry filter paper, the membrane was placed on a filter paper previously soaked with 2 x SSCP/0.2 M Tris-HCl, pH 7.4 and air-dried on a dry filter paper. The same procedures were repeated.
  • the treated nitrocellulose membranes were heated at 80°C for 2 hr to fix nucleic acid and washed twice with 3 x SSC (20 x SSC, i.e., SSC solution of 20 times of the concentration, consists of 3 M sodium chloride, 0.3 M sodium citrate)/0.1% SDS at 60°C for 15 min.
  • 3 x SSC i.e., SSC solution of 20 times of the concentration, consists of 3 M sodium chloride, 0.3 M sodium citrate)/0.1% SDS at 60°C for 15 min.
  • Hybridization was performed by incubating the membranes in a hybridization buffer containing 32 P-labeled DNA fragment previously prepared in Example 1 at a concentration of 5 ng/ml (converted to a template DNA basis) at 55°C for 18 hr.
  • the membranes were removed and washed in 3 x SSC/0.1% SDS at room temperature for 30 min, which was repeated twice.
  • the membranes were washed in 0.2 x SSC/0.1% SDS at 55°C for 15 min, which was repeated twice, dried and detected by autoradiography.
  • autography there obtained one positive plaque which gave positive signal at corresponding positions on both of paired membranes.
  • Clones corresponding to positive signal were recovered by punching the plaque on soft-agar with a glass tube and extracted with TMG buffer (50 mM Tris-HCl, pH 7.5, 100 mM sodium chloride, 10 mM magnesium chloride, 0.01% gelatin) (1 ml) in the presence of chloroform (50 ⁇ l) overnight.
  • TMG buffer 50 mM Tris-HCl, pH 7.5, 100 mM sodium chloride, 10 mM magnesium chloride, 0.01% gelatin
  • the extracted phage particles were subjected to plaque hybridization in a manner similar to that described above by transfecting into E. coli strain P2-392 in a conventional manner and culturing in 9 cm petri dishes in an appropriate amount. This series of procedures were repeated to purify the clone corresponding to the positive signal, resulting in an independent ⁇ 411 clone.
  • DNA fragments were extracted from ⁇ 114 phage clones obtained in Example 2 and subcloned into plasmids pUC18 and pUC 19.
  • a suspension of ⁇ phage clones (2 x 10 7 p.f.u., plaque formation unit) in TMG buffer (200 ⁇ l) were infected into E . coli strain P2-392 (2 x 10 8 , 40 ⁇ l) in NZY medium (200 ml) in 500 ml Erlenmeyer flask at 37°C for 15 min. After addition of 1 M calcium chloride (1 ml), the flask was incubated overnight, i.e., about 14 hr.
  • the mixture was centrifuged at 6,000 rpm for 20 min with Hitachi Cooling Centrifuge SCR20BB (Rotor RPR9-2) and the precipitates suspended in A buffer (0.5% NP40, 36 mM calcium chloride, 30 mM Tris-HCl, pH 7.5, 50 mM magnesium chloride, 125 mM potassium chloride, 0.5 mM EDTA, 0.25% DOC, 0.6 mM mercaptoethanol) (6 ml).
  • a buffer 0.5% NP40, 36 mM calcium chloride, 30 mM Tris-HCl, pH 7.5, 50 mM magnesium chloride, 125 mM potassium chloride, 0.5 mM EDTA, 0.25% DOC, 0.6 mM mercaptoethanol
  • coli were decomposed by incubating the suspension in the presence of 10 mg/ml deoxyribonuclease I (100 ⁇ l) and 10 mg/ml ribonuclease A (10 ⁇ l) at 30°C for 30 min.
  • To the reaction mixture is added an equal amount of chloroform and the mixture stirred thoroughly and centrifuged with Tomy Centrifuge LC-06 (Rotor-TS-7) at 3,000 rpm for 10 min to separate supernatant.
  • the nuclease-treated phage suspension prepared above was layered over these glycerol solutions and centrifuged with Hitachi Ultracentrifuge 70P72 (Rotor RPS40T; Hitachi, Japan) at 35,000 rpm for 1 hr.
  • Precipitated phage particles were suspended in a mixture (0.4 ml) of 40 mM Tris-HCl, pH 7.5, 10 mM EDTA and 2% SDS and the suspension incubated at 55°C for 1 hr in the presence of 10 mg/ml proteinase K (4 ⁇ l).
  • the solution was transferred to Eppendorph tube and extracted with an equal volume of phenol/chloroform.
  • the extract was subjected to ethanol precipitation to yield objective phage DNA (200 ⁇ g).
  • the resultant phage DNAs (10 ⁇ g) were digested with restriction enzymes Bam HI and Sal I (20 units each; Takara Syuzo, Japan) in a restriction buffer as specified in manual at 37°C for 3 hr and the digestion mixture was electrophoresed on agarose gel in a conventional manner.
  • the objective DNA fragment was separated and purified by digesting phage DNAs (100 ⁇ g) with restriction enzymes Bam HI and Sal I and recovering DNA fragment from agarose gel in a conventional manner.
  • the DNA fragments (about 100 ng) were ligated into pUC18 and pUC19 plasmid vectors (200 ng) previously digested with restriction enzymes Bam HI and Sal I in a conventional manner in the presence of T4 DNA ligase (350 units) in a reaction buffer (66 mM tris-HCl (pH 7.6), 6.6 mM magnesium chloride, 10 mM dithiothreitol, 66 ⁇ M ATP, substrate DNA) (10 ⁇ l).
  • the ligation mixture (1 ⁇ l) was used to transform competent E. coli DH5 ⁇ (COMPETENT HIGH, Toyobo, Japan) and the resultant bacterial solution was spread onto LB agar medium (1% yeast extract, 0.5% bacto-trypton, 0.5% sodium chloride) containing ampicillin (50 ⁇ g/ml) in 15 cm petri dish. Ampicillin-resistant colonies appeared on dish (5 ml each) were grown. Plasmid DNAs were recovered according to the method of Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p.365-370 (1982 ), digested with restriction enzymes Bam HI and Sal I and analyzed on agarose gel electrophoresis. The analysis revealed that a recombinant named p411BS containing the objective 5.6 kb DNA fragment was obtained.
  • Plasmid DNA was prepared from one of positive clones in a conventional manner ( Molecular Cloning, Cold Spring Harbor Laboratory, p.86-96 (1982 )) and DNA regions necessary for sequencing were digested with a restriction enzyme(s) into fragments, which were subcloned into plasmid vector pUC19. From the resultant subclones were prepared plasmid DNAs in a conventional manner and subjected to sequence analysis. Both (+)- and (-)-strands of the DNA fragment were determined using as sequence primers two synthetic primers shown below and in SEQ ID NO: 9 and 10, respectively. SEQ ID NO: 9: 5'-d(GTAAAACGACGGCCAGT) 3' SEQ ID NO: 10: 5'-d(CAGGAAACAGCTATGAC) 3'
  • plasmid p411BS derived from independent ⁇ 411 clone contains 5' upstream sequence, that is, a part of coding region (exon) corresponding to N-terminal region (amino acid No. 1 - 72) of hChM-I as well as intron sequence(s).
  • cDNA was carried out using a primer prepared by heating a mixture of 1.2 ⁇ g/ ⁇ l of a synthetic DNA (e.g., a DNA of SEQ ID NO: 11) containing Not I and, at its 5' end, Xho I restriction sites, and 40 Ts at the 3' end of NotI restriction site of one strand, which is shown by the following sequence: 5'-d(CTCGAGGCCATGGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • RNA was prepared as follows. Human chondrosarcoma tissue (10 g) is pulverized in liquid nitrogen, homogenize in aqueous guanidium isothiocyatate solution, and subjected to cesium chloride equilibrium density gradient centrifugation according to the method of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979) to obtain total RNA (about 1 mg). The total RNA is then purified using oligo(dT)cellulose type 7 (Pharmacia) according to a conventional method ( Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 85 (1982 )) to obtain polyA + RNA).
  • oligo(dT)cellulose type 7 Pharmacia
  • the polyA + RNA (about 1 ⁇ g) was reacted with the primer (80 pmole) previously prepared by annealing in a reaction buffer [50 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 8 mM magnesium chloride, 1 mM 4dNTPs (dATP, dGTP, dCTP, dTTP), 10 mM DTT (dithiothreitol) and 40 ⁇ Ci ⁇ - 32 P-dCTP] (50 ⁇ l) in the presence of AMV reverse transcriptase (100 units) at 37°C to obtain the first strand cDNA hybridized with template RNA.
  • a reaction buffer 50 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 8 mM magnesium chloride, 1 mM 4dNTPs (dATP, dGTP, dCTP, dTTP), 10 mM DTT (dithiothreitol)
  • PCR was carried out with Perkin Elmer Cetus DNA Thermal Cycler using Gene Amp DNA Amplification Reagent Kit (Takara Syuzo, Japan) according to the manufacture's instruction.
  • a template DNA a reaction mixture (1 ⁇ l) of cDNA synthesis described in Example 4 was used.
  • x 10 reaction buffer 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.1%(w/v) gelatin
  • 10 ⁇ l 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.1%(w/v) gelatin
  • 10 ⁇ l 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.1%(w/v) gelatin
  • 10 ⁇ l 1.25 mM 4dNTPs (16 ⁇ l)
  • 20 ⁇ M primer #1 and #2 5 ⁇ l each
  • Tag DNA polymerase 0.5 ⁇
  • PCR was conducted by repeating 35 times of reaction cycle comprising the following steps: pre-treatment at 94°C for 10 min, denaturation at 94°C for 1 min, annealing at 44°C for 1.5 min and elongation at 72 °C for 2 min. The reaction was stopped by incubating at 72°C for 7 min.
  • Primers #1 and #2 are DNA primers of 19 and 16 nucleotides each being shown in SEQ ID NO: 13 and 14, respectively, having the following sequences. Primer #1: 5'-d(AGTCTCCAAGTGCCTCACT) 3' Primer #2: 5'-d(CGAGGCCATGGCGGCC) 3'.
  • the primer #1 is the upstream sequence of a gene designed on the basis of the human gene obtained in Example 3, while the primer #2 is a DNA fragment complementary to a part of the sequence shown in SEQ ID NO: 12 which has been used as a primer in the synthesis of cDNA.
  • primers #3 and #4 which are DNA primers of 18 nucleotides shown in SEQ ID NO: 15 and 16, respectively, and have the following sequence.
  • the primer #3 is a sequence corresponding to the beginning part of the region encoding hChM-I designed on the basis of the human gene obtained in Example 3, while the primer #4 is a sequence corresponding to the end part of a sequence encoding bovine chondromodulin-I protein.
  • the 2nd PCR was carried in a manner similar to that used in the 1st PCR, that is, using the same reaction buffer, dNTPs, enzyme treatment, in a reaction mixture adjusted to 100 ⁇ l with distilled water by repeating 35 times of reaction cycle comprising the following steps: pre-treatment at 94°C for 10 min, denaturation at 94°C for 1 min, annealing at 55°C for 1.5 min and elongation at 72°C for 2 min. The reaction was stopped by incubating at 72°C for 7 min.
  • the reaction mixture (10 ⁇ l from 100 ⁇ l) was analyzed by agarose gel electrophoresis and gel corresponding to about 1 kb band was cut out to recover DNA fragment in a conventional manner.
  • DNA recovered from the gel was extracted with phenol/chloroform (1:1), precipitated with ethanol and dissolved in sterilized deionized water (20 ⁇ l).
  • DNA is ligated into a cloning site of a commercially available pCR2 vector (Invitrogen, Inc.) by reacting the DNA solution (5 ⁇ l) obtained above with pCR2 vector (0.25 ⁇ g) in the presence of T4 DNA ligase (300 units) in a reaction buffer (66 mM tris-HCl (pH 7.6), 6.6 mM magnesium chloride, 10 mM dithiothreitol, 66 ⁇ M ATP, substrate DNA) at 16°C for 12 hr.
  • a reaction buffer 66 mM tris-HCl (pH 7.6), 6.6 mM magnesium chloride, 10 mM dithiothreitol, 66 ⁇ M ATP, substrate DNA
  • the ligation mixture (3 ⁇ l) was use to transform competent E. coli JM109 (COMPETENT HIGH, Toyobo, Japan) and the resultant bacterial solution was spread onto X-Gal-IPTG-LB agar medium (1% yeast extract, 0.5% bacto-trypton, 0.5% sodium chloride, 0.004% X-Gal, 1 mM IPTG) containing ampicillin (50 ⁇ g/ml) in 15 cm petri dish. Twelve colonies which are ampicillin resistant and do not show color development due to X-Gal were selected from colonies on the petri dish, and each (5 ml) was grown.
  • X-Gal-IPTG-LB agar medium 1% yeast extract, 0.5% bacto-trypton, 0.5% sodium chloride, 0.004% X-Gal, 1 mM IPTG
  • Plasmid DNA was then recovered according to the method of Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p.365-379 (1982 ), digested with restriction enzyme Eco RI and analyzed by means of electrophoresis on agarose gel. The analysis revealed that there obtained three transformants each containing plasmid DNA phCHM-13-6, phCHM-16-3 or phCHM16-5, which plasmid gives DNA fragment of different size upon Eco RI digestion.
  • Plasmid DNAs were prepared from each of purified positive clones, phCHM-13-6, phCHM-16-3 and phCHM16-5, in a conventional manner ( Molecular Cloning, Cold Spring Harbor Laboratory, p.86-96 (1982 )) and sequenced with Fluorescence Sequencer GENESIS 2.000 System. Both of (+)- and (-)-strand of the DNA were determined using as sequence primers two different synthetic primers shown in SEQ ID NO: 9 and 10, respectively.
  • SEQ ID NO: 9 5'-d(GTAA.AACGACGGCCAGT) 3'
  • SEQ ID NO: 10 5'-d(CAGGAAACAGCTATGAC) 3'
  • the sequence of 3'-downstream coding region of clones phCHM-13-6, phCHM-16-3 and phCHM16-5 obtained in Example 5 above corresponded to the DNA primer (5'-d(ACACCATGCCCAGGATGC) 3'; SEQ ID NO: 16), which is 18 nucleotides synthetic DNA based on bovine chondromodulin gene and was used in the 2nd PCR.
  • SEQ ID NO: 16 amino acid primer
  • RNA was prepared as follows. Human normal costochondral tissue (about 20 g) was pulverized in liquid nitrogen, homogenized in aqueous guanidium isothiocyatate solution, and subjected to cesium chloride equilibrium density gradient centrifugation according to the method of Chirgwin et al., Biochemistry, 18, 5294-5299 (1978) to obtain total RNA (about 2 mg).
  • RNA (20 ⁇ g) was then reacted with a primer (80 pmole) previously annealed in a manner similar to that used in Example 4 in reaction buffer [50 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 8 mM magnesium chloride, 1 mM 4dNTPs (dATP, dGTP, dCTP, dTTP), 10 mM dithiothreitol and 40 ⁇ Ci ⁇ - 32 P-dCTP] (50 ⁇ l) in the presence of AMV reverse transcriptase (100 units) at 37°C to obtain the first strand cDNA hybridized with template RNA. A portion of the resulting reaction solution was used as template in the PCR.
  • reaction buffer 50 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 8 mM magnesium chloride, 1 mM 4dNTPs (dATP, dGTP, dCTP, dTTP), 10 mM
  • PCR was carried out with Perkin Elmer Cetus DNA Thermal Cycler using Gene Amp DNA Amplification Reagent Kit (Takara Syuzo, Japan) according to the manufacture's instruction using, as template, a portion (1 ⁇ l) of the reaction solution obtained in the cDNA synthesis using total RNA as described above.
  • reaction buffer 500 Mm KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.1%(w/v) gelatin
  • 10 ⁇ l 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.1%(w/v) gelatin
  • 10 ⁇ l 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.1%(w/v) gelatin
  • 4dNTPs 16 ⁇ l
  • 20 ⁇ M primers #5 and #2 5 ⁇ l each
  • Taq DNA polymerase 0.5 ⁇ l
  • Primers #5 and #2 are DNA primers of 16 nucleotides each being shown in SEQ ID NO: 17 and 14, respectively, having the following sequence:
  • the reaction mixture (10 ⁇ l from 100 ⁇ l) was analyzed by agarose gel electrophoresis and a gel corresponding to about 0.6 kb band was cut out in a conventional manner to recover DNA fragment.
  • the DNA fragment was extracted with phenol/chloroform (1:1), precipitated with ethanol and dissolved in sterilized deionized water (20 ⁇ l).
  • the DNA was ligated into a cloning site of commercially available pCR2 vector (Invitrogene) by reacting the DNA solution (5 ⁇ l) obtained above with pCR2 vector (0.25 ⁇ g) (Invitrogen, Inc.) in the presence of T4 DNA ligase (350 units) in a reaction buffer (66 mM tris-HCl (pH 7.6), 6.6 mM magnesium chloride, 10 mM dithiothreitol, 66 ⁇ M ATP, substrate DNA) at 16°C for 12 hr.
  • the ligation mixture (3 ⁇ l) was used to transform competent E. coli JM109 (COMPETENT HIGH, Toyobo, Japan) and the resultant bacterial solution was spread onto X-Gal-IPTG-LB agar medium (1% yeast extract, 0.5% bacto-trypton, 0.5% sodium chloride, 0.004% X-Gal, 1 mM IPTG) containing ampicillin (50 ⁇ g/ml) in 15 cm petri dish. Twelve colonies which are ampicillin resistant and do not show color development due to X-Gal were selected from colonies appeared on the petri dish, and each (5 ml) was grown.
  • X-Gal-IPTG-LB agar medium 1% yeast extract, 0.5% bacto-trypton, 0.5% sodium chloride, 0.004% X-Gal, 1 mM IPTG
  • Plasmid DNA was then recovered according to the method of Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p.365-370 (1982 ), digested with restriction enzyme Eco RI and analyzed by electrophoresis on agarose gel. The analysis revealed that there obtained two transformants, phCHM-ILAST8 and phCHM-ILAST12, which give about 0.6 kb DNA fragments other than vector DNA fragments upon digestion with restriction enzyme Eco RI. 16-3 or phCHM16-5, which give DNA fragment of different size upon Eco RI digestion.
  • Plasmid DNAs were prepared from both purified clones phCHM-ILAST8 and phCHM-ILAST12 in a conventional manner ( Molecular Cloning, Cold Spring Harbor Laboratory, p.86-96 (1982 )) and sequenced with Fluorescence Sequencer GENESIS 2,000 System (Dupont). Both of (+)- and (-)-strand of the DNA were determined using as sequence primers two different synthetic primers shown in SEQ ID NO: 9 and 10, respectively.
  • SEQ ID NO: 9 5'-d(GTAAAACGACGGCCAGT) 3'
  • SEQ ID NO: 10 5'-d(CAGGAAACAGCTATGAC) 3'
  • Plasmid DNA was obtained from cDNA clones phCHM-13-6, phCHM-16-3 or phCHM16-5 prepared in Example 5 according to the method of Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p. 86-96 (1982 ).
  • Each of plasmid DNAs phCHM-13-6, phCHM-16-3 and phCHM16-5 was digested with restriction enzymes Not I and Nsi I to obtain about 1 kb DNA fragment containing part of vector sequence from respective clones. These DNA fragments covered the whole region encoding hChM-I, that is, the region extending from the translation initiation codon ATG through the stop codon TAA.
  • Each of the DNA fragments recovered from cDNA clones phCHM-13-6, phCHM-16-3 and phCHM16-5 was ligated into commercially available expression vector pcDNAlneo (Invitrogen, Inc.) previously digested with restriction enzymes Not I and Nsi I in reaction system (10 ⁇ l) containing T4 DNA ligase in a conventional manner.
  • the ligation mixture was used to transform competent E . coli DH5 ⁇ (COMPETENT HIGH, Toyobo, Japan) and the resultant bacterial solution was spread onto LB agar medium (1% yeast extract, 0.5% bacto-trypton, 0.5% sodium chloride) containing ampicillin (50 ⁇ g/ml) in 15 cm petri dish.
  • Plasmid DNAs were recovered according to the method of Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p.365-370 (1982 ), digested with restriction enzymes Not I and Nsi I and analyzed on agarose gel electrophoresis. The analysis revealed that desired recombinants transformed with plasmid DNAs pcDNAhCHM-13-6, pcDNAhChM-16-3 and pcDNAhChM16-5 each containing, at the restriction sites Not I and Nsi I of expression vector pcDNAineo, each of objective hChM-I cDNA fragment were obtained.
  • E . coli cells were recovered and purified plasmid DNAs according to the method of Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p.86-96, (1982 ) to obtain a large amount of hChM-I expression plasmid DNAs.
  • COS cells were transfected with either of expression plasmids pcDNAhCHM-13-6, pcDNAhChM-16-3 and pcDNAhChMl6-5 constructed in Example 6 by the use of commercially available lipofectin reagent (LIPOFECTIN TM , GIBCO, Inc.) according to the manufacture's instruction.
  • lipofectin reagent LIPOFECTIN TM , GIBCO, Inc.
  • COS cells were grown in DMEM medium in 9 cm petri dish. After removal of medium, cells were washed twice with PBS(-) solution (0.8% sodium chloride, 0,02% potassium chloride, 0.144% disodium hydrogen phosphate, 0.024% sodium dihydrogen phosphate, pH 7.4). After removal of PBS(-) solution, serum-free DMEM medium (8 ml) was added to the plate.
  • a DNA solution to be used in transection has been prepared by dissolving plasmid pcDNAhCHM-13-6, pcDNAhChM-16-3 or pcDNAhChM16-5 DNA (20 ⁇ g) in serum-free DMEM medium (100 ⁇ l) in No.
  • FCS-containing ERDF medium (Kyokuto-seiyaku, Inc.) was added to 10 cm petri dish and incubation continued at 37°C for about 56 hr under an atmosphere of 5% CO 2 .
  • Cultured broth was collected and the presence of physiological activity of hChM-I was confirmed according to a known method ( Suzuki, et al., Methods in Enzymology, 146: 313-320 (1987) ). The expression of hChM-I was confirmed by Western blotting conventionally.
  • the cell suspension (0.1 ml) was dispersed into 96-well plate, which had been coated with type I collagen (50 ⁇ g/ml)overnight and washed with FAD medium, and incubated at 37 °C under atmosphere of 5% CO 2 with changing the medium every other day.
  • the DNA-synthetic activity was evaluated as follows. Cells were grown in the above 96-well plate until the culture became confluent, then the cells were grown in FAD medium containing 0.3% FCS for 24 hr. The culture was incubated for 22 hr in 0.1 ml of FAD medium containing 0.06 to 20 ng of cultured broth of transformants containing hChM-I, 0.04 ng of FGF (fibroblast growth factor) and 0.3% FCS.
  • the cultivation was continued another 4 hr after the addition of 10 ⁇ l of [ 3 H]thymidine (130 ⁇ Ci/ml) and cells were washed three times with ice-cold phosphate-buffered saline (20 mM phosphate buffer, pH7.0, 0.15 M sodium chloride), extracted with 5% trichloroacetic acid and then with ethanol/ether (3:1 in volume). After the extraction, the precipitate left was dissolved in 0.3 M sodium hydroxide, neutralized with 1/20 volume of 6N HCl and the radioactivity was detected by means of a scintillation counter.
  • bovine aortic endotheliocytes were inoculated into ⁇ -MEM medium containing 0.1 ml of 20% FCS in a 96-well plate at a cell density of 2 x 10 3 cells/well and grown in a CO 2 incubator at 37°C for 48 hr, when the medium was replaced by a freshly prepared one and 0 to 3 ⁇ g/ml of cultured broth of transformants containing hChM-I added thereto.
  • hChM-I of the present invention is novel protein originated from human. By the use of gene encoding hChM-I, one can provide sufficient amount of recombinant hChM-I constantly and steadily.
  • the hChM-I has activities to stimulate the growth of chondrocyte, to promote the differential potency of chondrocyte and to inhibit the growth of endothelial cells, and can be useful as an active ingredient of medicinal drugs.
  • the hChM-I of the present invention is distinct from conventional bovine chondromodulin protein in amino acid sequence, base sequence of gene encoding the same and the like, and is considered to be less antigenic and more effective when used in treatment of human.

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Claims (12)

  1. Menschliches Chondromodulin-I-Protein, umfassend eine Aminosäuresequenz dargestellt in SEQ ID NO: 2, 3 oder 4, mit der Fähigkeit, das Wachstum von Chondrozyten zu stimulieren.
  2. Isolierte DNA, die das menschliche Chondromodulin-I-Protein von Anspruch 1 codiert.
  3. DNA gemäß Anspruch 2, wobei das DNA-Molekül die in SEQ ID NO: 2 bis 7 dargestellte Nukleotidsequenz umfaßt.
  4. Expressionsvektor, der das Gen gemäß den Ansprüchen 2 und 3 exprimieren kann.
  5. Transformante, transformiert mit einem Expressionsvektor gemäß Anspruch 4.
  6. Verfahren zur Erzeugung des Chondromodulin-I-Proteins, das das Kultivieren der Transformante von Anspruch 5 in einem geeigneten Medium für die Expression der DNA, codierend das Chondromodulin-I-Protein, und das Gewinnen des Chondromodulin-I-Proteins aus der resultierenden kultivierten Brühe umfaßt.
  7. Rekombinantes menschliches Chondromodulin-I-Protein, umfassend eine Aminosäuresequenz wie dargestellt in SEQ ID NO: 2, 3 oder 4.
  8. Pharmazeutische Zusammensetzung, enthaltend als Wirkstoff eine effektive Menge eines Chondromodulin-I-Proteins gemäß Anspruch 1 zusammen mit einem pharmazeutisch annehmbaren Träger, Exzipienten oder Lösungsmittel.
  9. Pharmazeutische Zusammensetzung gemäß Anspruch 8, die zur Stimulation des Wachstums von Chondrozyten verwendet wird.
  10. Pharmazeutische Zusammensetzung gemäß Anspruch 8, wobei es sich um ein Antitumorarzneimittel handelt.
  11. Verwendung einer pharmazeutischen Zusammensetzung gemäß Anspruch 8 für die Herstellung eines Medikaments zur Stimulation des Wachstums von Chondrozyten.
  12. Verwendung einer pharmazeutischen Zusammensetzung gemäß Anspruch 8 für die Herstellung eines Medikaments zur Behandlung von Krebs.
EP94107364A 1993-05-11 1994-05-11 Menschliches Chondromodulin-I-Protein Expired - Lifetime EP0624645B1 (de)

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US6140305A (en) * 1996-04-04 2000-10-31 Bio-Rad Laboratories, Inc. Hereditary hemochromatosis gene products
US7026116B1 (en) * 1996-04-04 2006-04-11 Bio-Rad Laboratories, Inc. Polymorphisms in the region of the human hemochromatosis gene
WO1997039116A1 (en) * 1996-04-12 1997-10-23 Novo Nordisk A/S Enzyme-containing granules and process for the production thereof
US6849399B1 (en) 1996-05-23 2005-02-01 Bio-Rad Laboratories, Inc. Methods and compositions for diagnosis and treatment of iron misregulation diseases
DE69841593D1 (de) 1997-06-13 2010-05-12 Bio Rad Laboratories Verfahren und Zusammensetzungen für die Diagnose und die Behandlung von Krankheiten, die mit Eisenüberschuss oder Eisenmangel assoziiert sind
JP2002534972A (ja) * 1999-01-19 2002-10-22 ヒューマン ジノーム サイエンシーズ， インコーポレイテッド ３３個のヒト分泌タンパク質
EP1219710B1 (de) 1999-09-29 2010-04-07 Teijin Limited POLYPEPTIDE UND FüR DIESE KODIERENDE GENE
ATE286911T1 (de) * 1999-11-26 2005-01-15 Takeda Pharmaceutical Transkriptionsfaktor und dessen dna
EP1374888A4 (de) * 2001-02-28 2004-07-07 Mitsubishi Pharma Corp Heilmittel gegen arthritis deformans und heilmittel gegen rheumatoide arthritis
CN1592635A (zh) * 2001-09-24 2005-03-09 维里根股份公司 自体生长因子鸡尾酒组合物,生产方法以及应用
US8697139B2 (en) 2004-09-21 2014-04-15 Frank M. Phillips Method of intervertebral disc treatment using articular chondrocyte cells
US20100145441A1 (en) * 2005-09-22 2010-06-10 Keiichi Fukuda Therapeutic agents for angiogenesis-related diseases comprising chondromodulin-i as active ingredient

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US5444157A (en) * 1990-08-23 1995-08-22 Mitsubishi Kasei Corporation Chondromodulin-I protein
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